Registration Dossier

Administrative data

Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was conducted according to OECD and EU methods and under GLP conditions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
-Chemical Name: 6-hydroxy-2-naphthaleneboronic acid
- Substance type: off white powder
- Physical state: solid
- Storage condition of test material: room temperatire until 14 October 2013 and then approximately 4° C in the dark
- Date received: 08 October 2013

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd, Oxon, UK
- Age at study initiation: approx 6 -8 weeks old
- Weight at study initiation: males weighed between 194-232g; females 146 to 178g
- Housing: The animals were housed in groups of five by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK)
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 50 ± 20%
- Air changes: at least 15 per hour
- Photoperiod: 12 hrs dark / 12 hrs light):

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test item was prepared at the appropriate concentrations as a suspension in 1% Carboxy methylcellulose. The stability and homogeneity of the test item formulations were determined by Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. Results show the formulations to be stable for at least twenty one days. Formulations were therefore prepared twice during the treatment period and stored at approximately 4 ºC in the dark.

Samples of each test item formulation were taken and analyzed for concentration of Boronic Acid at Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. The method used for analysis of formulations and the results obtained are given in Appendix 18. The results indicate
that the prepared formulations were within ± 7% of the nominal concentration.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test item concentration in the test samples was determined by high performance liquid chromotography with UV detection (HPLC/UV) using an external standard technique. The test item gave a chromatogrphic profile consisting of a single peak.
The detection system was found to have acceptable linearity. The analytical procedure had acceptable recoveries of test item in vehicle. The method of analysis was validated and proven to be suitable for use.
Duration of treatment / exposure:
28 days
Frequency of treatment:
The test item was administered daily, for up to twenty-eight consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 5 mL/kg of 1% Carboxy methylcellulose.
The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 20, 300, 750 MG/KG BW/DAY
Basis:
actual ingested
No. of animals per sex per dose:
5 male, 5 female
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were chosen based on the resultsts of previous toxicity work.
The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item, and the results of the study are believed to be of value in predicting the likely toxicity of the test item to man.

- Rationale for animal assignment: The rat was selected for this study as it is a readily available rodent species historically used in
safety evaluation studies and is acceptable to appropriate regulatory authorities.
- Section schedule rationale: The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups.
Positive control:
not applicable

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Prior to the start of treatment and on Days 5, 14, 21 and 27, all animals were observed for signs
of functional/behavioral toxicity. Functional performance tests were also performed on all animals during Week 4, together with an assessment of sensory reactivity to different stimuli. Observations were carried out from approximately two hours after dosing on each occasion.

- Cage side observations below were included:
Gait Hyper/Hypothermia
Tremors Skin color
Twitches Respiration
Convulsions Palpebral closure
Bizarre/Abnormal/Stereotypic behavior Urination
Salivation Defecation
Pilo-erection Transfer arousal
Exophthalmia Tail elevation
Lachrymation

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health or behavioral change immediately before dosing, up to thirty minutes post dosing and one hour after dosing throughout the study period. All observations were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Day 1 and at weekly intervals thereafter. Body weights were also performed prior to terminal kill.

FOOD CONSUMPTION: YES
- Food consumption was recorded for each cage group at weekly intervals throughout the study. Food conversion efficiency was calculated retrospectively.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Water intake was measured and recorded daily for each cage group.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Hematological and blood chemical investigations were performed on all animals from each test and control group at the end of the study (Day 28).
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: all animals i.e. 40
- Parameters listed below were examined:
Hemoglobin (Hb)
Erythrocyte count (RBC)
Hematocrit (Hct)
Erythrocyte indices - mean corpuscular hemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular hemoglobin concentration (MCHC)
Total leukocyte count (WBC)
Differential leukocyte count - neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic)


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Hematological and blood chemical investigations were performed on all animals from each test and control group at the end of the study (Day 28).
- Animals fasted: No
- How many animals: all animals i.e. 40
- Parameters checked
Blood Urea Inorganic phosphorus (P)
Glucose Aspartate aminotransferase (ASAT)
Total protein (Tot.Prot.) Alanine aminotransferase (ALAT)
Albumin Alkaline phosphatase (AP)
Albumin/Globulin (A/G) ratio (by calculation) Creatinine (Creat)
Sodium (Na+) Total cholesterol (Chol)
Potassium (K+) Total bilirubin (Bili)
Chloride (Cl-) Bile acids
Calcium (Ca++) Triglycerides

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to the start of treatment and on Days 5, 14, 21 and 27, all animals were observed for signs of functional/behavioral toxicity. Functional performance tests were also performed on all animals during Week 4, together with an assessment of sensory reactivity to different stimuli. Observations were carried out from approximately two hours after dosing on each occasion.
- Dose groups that were examined: all animals
- Battery of functions tested:
Motor activity: Twenty infra-red beam automated activity monitors were used to assess motor activity. Animals of one sex were tested at each occasion and were randomly allocated to the activity monitors. The tests were performed at approximately the same time each
occasion (at least two hours after dosing), under similar laboratory conditions. The evaluation period was one hour for each animal. The time in seconds each animal was active and mobile was recorded for the overall one hour period and also during the final 20% of the period (considered to be the asymptotic period, Reiter and Macphail 1979).
Sensory activity: Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. This assessment was developed from the methods employed by Irwin (1968) and Moser et al (1988). The scoring system used is outlined in attachement below entitled "Key to Scoring System and Explanation for Behavioral Assessments and Sensory Reactivity Tests"

The following parameters were observed:
Grasp response Touch escape
Vocalization Pupil reflex
Toe pinch Blink reflex
Tail pinch Startle reflex
Finger approach
Forelimb/hindlimb grip strength;
OTHER:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table attached below)
HISTOPATHOLOGY: Yes (see table attached below)

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
SEE BELOW
Mortality:
mortality observed, treatment-related
Description (incidence):
SEE BELOW
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
SEE BELOW
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
SEE BELOW
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
SEE BELOW
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
SEE BELOW
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS
At 750 mg/kg bw/day, all males and three females showed episodes of increased salivation. At 300 mg/kg bw/day one male and four females showed isolated episodes of increased salivation.
No such effects were detected in animals of either sex treated with 30 mg/kg bw/day.
MORTALITY
There were no unscheduled deaths.

BODY WEIGHT AND WEIGHT GAIN
Animals of either sex treated with 750 mg/kg bw/day showed a statistically significant reduction in body weight gain during the first week of treatment. Overall body weight gain was reduced by approximately 30% for animals of either sex.
No adverse effect of treatment was detected in animals of either sex treated with 300 or 30 mg/kg bw/day.

FOOD CONSUMPTION AND FOOD EFFICIENCY
Animals of either sex treated with 750 mg/kg bw/day showed a slight reduction in food consumption during Week 1 of the study when compared to controls. Animals of either sex treated with 750 mg/kg bw/day also showed a reduction in food conversion efficiency during Weeks 1 and 3. Males treated with 750 mg/kg bw/day also showed a slight reduction in food conversion efficiency during Week 4.

WATER CONSUMPTION
Animals of either sex treated with 750 mg/kg bw/day showed an approximate two fold increase in overall water consumption when compared to controls.
Females treated with 300 mg/kg bw/day showed a noteworthy increase in overall water consumption.
No such effects were detected in males treated with 300 mg/kg bw/day or animals of either sex treated with 30 mg/kg bw/day.

HAEMATOLOGY
No toxicologically significant effects were detected in the hematological parameters examined.

BLOOD CHEMISTRY
No toxicologically significant effects were detected in the blood chemical parameters examined.

BEHAVIOURAL ASSESSMENT
There were no toxicologically significant changes in the behavioural parameters measured.

FUNCTIONAL PERFORMANCE TESTS
There were no toxicologically significant changes in functional performance.

SENSORY REACTIVITY ASSESSMENTS
There were no treatment-related changes in sensory reactivity.

ORGAN WEIGHTS
There were no toxicologically significant changes in organ weights.

HISTOPATHOLOGY: NON-NEOPLASTIC
The following treatment related microscopic abnormalities were detected:
Mucosal alteration (minimal or mild) in the glandular stomach was present in 5/5 males and 2/5
females treated with 750 mg/kg bw/day and 2/5 males treated with 300 mg/kg bw/day (minimal).
This presented with minor disruption of the mucosa with some inflammatory cell infiltration,
mucosal expansion and foveolar hypertrophy with a cystic pattern. No changes were present in
animals of either sex treated with 30 mg/kg bw/day.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: systemic toxicity
Dose descriptor:
NOEL
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: systemic toxicity. The No Effect Level (NOEL) for systemic toxicity was considered to be 30 mg/kg bw/day.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

See file 'List of Tables' attached below

Applicant's summary and conclusion

Conclusions:
The oral administration of Boronic Acid to rats by gavage for a period of twenty eight
consecutive days at dose levels of 30, 300 and 750 mg/kg bw/day resulted in treatment related
effects in animals of either sex treated with 300 or 750 mg/kg bw/day.

The effects detected at 750 mg/kg bw/day were sufficient in severity to preclude this dose level
as a No Observed Adverse Effect Level (NOAEL), however the effects detected at 300 mg/kg
bw/day were less severe and in the absence of any effect on body weight, 300 mg/kg bw/day was
considered to be the No Observed Adverse Effect Level (NOAEL) for systemic toxicity. The No Effect Level (NOEL) for systemic toxicity was considered to be 30 mg/kg bw/day.
Executive summary:

The study was performed between 13 November 2013 and 22 July 2014 (date of final histopathology report). The in-life phase of the study was conducted between 20 December 2013 (first day of treatment) and 17 January 2014 (final day of necropsy).

The study was designed to investigate the systemic toxicity of the test item, Boronic Acid. The test item was administered by oral gavage to three groups, each of five male and five female Wistar Han strain rats, for 28 consecutive days, at dose levels of 30, 300 and 750 mg/kg bw/day. A countrol group of five males and five females were dosed with vehicle alone (1% Carboxy methylcellulose).

Clinical signs, functional observations, body weight change, dietary intake and water consumption were monitored during the study. Hematology and blood chemistry were evaluated for all animals at the end of the study.

All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues was performed